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In principle, lactic acid bacteria (LAB) and many other bacteria and generally most microorganisms, do not have it easy to survive in beer or other alcoholic beverages such as wine. This is one of the main reasons why wines and beers have been from ancient times the safest ways to drink hygienically something similar to water and that it was not contaminated, apart from boiled waters, such as tea and other herbal infusions.

The reasons for the difficult survival of microorganisms in beer are ethanol, the pH quite acidic (around 4), the lack of nutrients due to the fact that the yeasts have assimilated them, the little dissolved oxygen, the high concentration of carbon dioxide (0.5% by weight / volume) and the presence of humulone derived compounds (Figure 1) of hops: iso-alpha-acids, up to 50 ppm, which are microbiocides. All these obstacles make it very difficult for any microorganism to thrive. The most susceptible beers of unwanted microbial growth are those where some of the mentioned obstacles are dampened: beers with a higher pH of 4.5, or with little ethanol or little CO2, or with added sugars – which are nutrients -, or with little amount of compounds derived from hops (Vriesekoop et al 2012).

Figure 1. Humulone (left) of the hop is degraded during beer elaboration to isohumulone (right) and other iso-alpha-acids, which are compounds bitter and microbiocides (Wikipedia; Sakamoto & Konings 2003)

The acid pH of the beer (slightly higher than the wine) inhibits many of the best-known pathogens (Figure 2). And the cases we see that could grow at this pH near 4 are inhibited by other factors such as ethanol.

Figure 2. Range of acid pH for the growth of various bacteria, compared to the typical beer pH (Menz et al 2009).

The “bad” lactic acid bacteria of beer

Despite what we have just seen, some bacteria, particularly some LAB, have been able to adapt evolutionarily to the strict beer conditions, and they can survive and spoil them. In particular, the most frequent harmful species against the quality of beers are Lactobacillus brevis and Pediococcus damnosus (Figure 3). The first is the most frequent, and it can give tastes and undesired aromas, as well as turbidity to the final product. P. damnosus has the advantage of growing at low temperatures, and it can also produce undesired aromas, such as diacetyl (Vriesekoop et al 2012). Some Pediococcus and Lactobacillus may adhere to yeast, inducing them to sediment, which delays fermentation (Suzuki 2011).

Some Pediococcus may also be responsible for the appearance of biological amines in some beers, at risk for the consumer. Amines in a certain concentration are toxic, they may be present in some fermented foods such as cheese, cold meat and alcoholic beverages such as wines and beers, and are produced by decarboxylation of amino acids by LAB. The level of tyramine and other amines has been used as a measure of quality in some Belgian beers made with LAB (Loret et al 2005).

Apart from these LAB, other bacteria related to problems of beer contamination are acetic acid bacteria such as Acetobacter, typically associated with oxygen intake in packaging or distribution. Other harmful bacteria are some enterobacteria, such as Shimwellia pseudoproteus or Citrobacter freundii, which proliferate in the early stages of fermentation, and produce butanediol, acetaldehyde and other unwanted aromatic compounds (Vriesekoop et al 2012). Other harmful bacteria for beer, especially when bottled, are Pectinatus and Megasphaera, which are strict anaerobes, of the clostridial family, and can produce hydrogen sulphide and short chain fatty acids, all of them unpleasant (Suzuki 2011 ).

The “good” lactic acid bacteria of beer

LAB are well known for being some of the microbes that most benefits contribute to the food production, on the one hand as an economic means of preserving food, and on the other hand to improve their quality and organoleptic characteristics. That’s why they are the main agents of fermented foods, along with yeasts. We have seen some of the LAB’s food benefits in other posts in this blog: prehistoric cheeses, or breast milk microbiota, and even wine bacteria.

Therefore, LAB also have a good role in the production of beers: in particular, as we will see below, in the production of acidified malt, and in some peculiar styles of beer such as the Belgian Lambic and the Berliner Weissbier.

As you know, malt is the raw material for making beer. The cereal is subjected to the malting process, where cereal grains, mainly barley, are germinated, the enzymes hydrolyse the starch into sugars, and all of this is then heated obtaining the must, the substrate solution which will be fermented by the yeasts ferment, producing ethanol and carbon dioxide.

The acidification of the malt, that is, with a lower pH, has the advantages of activating many important enzymes in malting, giving a lower viscosity to the malt and therefore to the final beer. Although adding mineral acids or commercial lactic acid can achieve acidification, it is often recommended or legislated a biological acidification, which is achieved by adding LAB. The use of LAB starter cultures is a relatively new process and in addition to the commented benefits on the quality of the malt, it has been shown to also inhibit unwanted molds that are a real problem in malting and that can give mycotoxins. The compounds produced by LAB that can inhibit the fungi are the same lactic acid and the consequent pH drop, bacteriocins, hydrogen peroxide, and other compounds not well known as perhaps some peptides (Lowe & Arendt 2004).

The most commonly LAB strains used to acidify malt are Lactobacillus amylolyticus previously isolated from the same malt. These strains are moderately thermophile, resistant to compounds derived from humulone, and they have the advantage of being amylolytic in addition to producing lactic acid, which lowers the pH (Vriersekoop et al 2012).

Beers with LAB participating in the fermentation, such as Lambic and Berliner Weissbier styles, belong to the type of spontaneous fermentation beers. The other types of controlled fermentation beers are the best-known Ale and Lager, both inoculated with specific yeasts. Ale beers are those of high fermentation, where Saccharomyces cerevisiae yeast used tends to remain on the surface and the fermentation temperature is above 15-20ºC. Lager ones are those of low fermentation, originally from Bavaria, where yeast S. pastorianus (S. carlsbergensis) tends to settle at the bottom of the fermenter and the temperature is between 7 and 13ºC.

Belgian Lambic beer

Traditional Belgian beers (in Dutch lambiek or lambik) are known for their sensorial characteristics due to LAB activity. They are traditional in Brussels itself and in the neighbouring region of Pajottenland, in the Zenne river valley, in the Flemish Brabant on the SW of the Belgian capital. One of the villages in this valley is Lembeek, which could be the origin of the name of this beer.

These beers of spontaneous fermentation represent the oldest style of making beer in the developed world, for some centuries. For a few years now (since around 2008), similar beers are made in the USA, called “American coolship ales” (Ray 2014).

Lambic beer is made with barley malt and a minimum of 30% of non-malted wheat. The cones of a special hops, completely dried and aged for 3 years, are added to the must. They are added not for their aroma or bitterness, but rather as antimicrobial, to prevent above all, the growth of gram-positive pathogenic bacteria in the fermentation broth.

Also to avoid these contaminants and to promote the microbiota typical of the Lambic fermentation, these beers are brewed only between October and May, since in summer there are too many harmful microorganisms in the air that could spoil the beer, and it is necessary to lower the temperature after boiling. Boiling of the must is done intensively, with an evaporation of 30%.

After boiling, the broth is left in open deposits, and in this way the microorganisms of the air present in the fermentation rooms of the brewery (usually at the top of the building) are acquired, and of the outside air, since the tradition says that the windows must be left open. It is assumed that the captured microbes are specific to the Zenne Valley. These open deposits are the koelschip in Dutch (coolship in English), like swimming pools (Figure 4). Being well open, with a lot of surface (about 6 x 6 m) and shallow depth (about 50 cm), they favour the collection of microbes from the room and from the outside. Another purpose of this form is the fastest cooling of boiled broth to start fermentation. They can be made of wood, copper, or stainless steel more recently.

Figure 4. Koelschip (in Dutch) or coolship in English, the open deposits, as swimming-pools, where the Lambic beer process begins (Brasserie Cantillon, Brussels).

The “inoculated” broth in this spontaneous way is left only one night in the coolship, and on the following day this must is pumped into fermentation tanks where there will stay a year, during which the sugar content will go down, up to about 30 g/L. Then it is transferred to oak barrels, previously used for sherry or port, and there it can be left for another two years, at temperatures of 15-25ºC. Some barrels are the same used since 100 years ago. The final product is a cloudy beer, with a pale yellow, very little CO2, dry, acidic, with about 6-8º of ethanol. It reminds a bit like the sherry and especially the cider, and with a slightly bitter taste (Jackson 1999).

In this long process of fermentation, up to 3 years, of course there is a diversity in the composition of the microbial population. In a first phase there is a certain predominance of Kloeckera yeasts and especially enterobacteria during the first month. After 2 months, Pediococcus damnosus and Saccharomyces spp. predominate, and alcoholic fermentation begins. After 6 months of fermentation the predominant yeast is Dekkera bruxellensis (Spitaels et al 2014), or what is the same, Brettanomyces (Kumara & Verachtert 1991), of which Dekkera is the sexual form.

Figure 5. Species of isolates in MRS and VRBG agar media, for lactic acid bacteria and enterobacteria respectively, during the process of making a Lambic beer. The number of isolates is given between brackets (Spitaels et al 2014).

We see (Figure 5) as in particular after 2 months the predominant bacterium is the LAB P. damnosus. It was appointed in the first studies as “P. cerevisiae“, but this name was finally not admitted because it included other species. The count of these in MRS is 104UFC per mL until the end of fermentation. Acidification seems to be rapidly taking place in the transition from the first stage to that of maturation, coinciding with the growth of P. damnosus, which produces lactic acid, although Dekkera/Brettanomyces and acetic acid bacteria also contribute to the acidification (Spitaels et to 2014).

In other trials with the American coolship ales (ACA) of Lambic style, Lactobacillus spp. have also been found, and in a metagenomic study (Bukolich et al 2012) of these ACA, DNA of several Lactobacillales has been detected. At the end of the process, a predominance of Pediococcus (Figure 6, panel C) was also observed. In the same figure in panel A we observe how the predominant unicellular fungus is also Dekkera/Brettanomyces.

The basic Lambic, which is difficult to purchase, is only found in a few Brussels cafes and the production area. In fact, Lambic is the basis for elaborating the others, much more common to consume:

The Faro is a Lambic sweetened with brown sugar and sometimes with spices.

The fruity Lambic are those that have been added whole fruits or fruit syrup. They can be with bitter cherry (kriek), which are the most traditional, or with raspberry, peach, grapes, strawberry, and sometimes also apple or pineapple or apricot or other.

And finally, the Gueuze, which are sparkling and easy to find. They are made by mixing young Lambics (from 6 months to 1 year) with other more mature ones (2-3 years) in thick glass bottles similar to those of champagne or cava and left for a second fermentation with the remaining sugars from the young Lambic. This would have been begun by a mayor of Lembeek in 1870 that owned a brewery and applied the fermentation techniques in the bottle that had been successful in the Champagne some years before (Cervesa en català 2012). The word Gueuze can have the same etymological origin as gist(yeast in Flemish) and it could also refer to the fact that it produces bubbles of CO2, that is, gas (Jackson 1999). However, another historical version would be that this beer was called “Lambic de chez le gueux” (Welsh from poor people) because the mentioned mayor of Lembeek had similar socialist ideas to those of the “Parti des Gueus” founded by the Calvinists from Flanders in the 16th century to fight against the Spanish empire. And since beer is feminine in French, the gueuxfeminine is gueuze, here it is.

In this refermentation in the bottle the populations of Dekkera/Brettanomycesand LAB are maintained, although other unicellular fungi such as Candida, Hansenula, Pichia or Cryptococcus (Verachtert & Debourg 1999) appear in limited numbers.

The Berliner Weissbier (Figure 8) is another beer relatively similar to Lambic ones. It is also brewed with an important part of wheat must, it is cloudy, acidic and with 3% ethanol. It is traditional in Berlin and the north of Germany, made from the s. XVI and the most popular alcoholic beverage in Berlin until the end of the s. XIX. It was called the “northern champagne” by the Napoleon’s soldiers. Spontaneous fermentation of must involves a mixture of Dekkera/Brettanomyces, Saccharomycesand hetero-fermentative Lactobacillus.

In the same way that the commented American Coolship Ales, Lambic style beers are also made in many other countries and, in the case of Spain, coinciding with the boom of artisanal beers, they are also elaborated, especially the fruity Lambic ones. According to the Birrapedia website, 6 of these are currently being processed, all of which are cherries. Two of them are made in Lleida, one in Barcelona, one in Alicante, one in the Jerte valley, and another in Asturias.

Resistance of lactic acid bacteria from beer to hop compounds

Lactobacillus and Pediococcus, both bad and good we have seen, and other contaminating bacteria of beers, have the ability to withstand hop compounds, which, as we have seen, are natural microbiocides. This resistance can be due to various defence systems, both active and passive (Sakamoto & Konings 2003). The active systems include efflux pumps, such as HorA and HorC, which carry the iso-alpha-acids (Figure 1) out the cell. HorA does it with ATP consumption, and HorC using the proton driving force (Figure 9). The corresponding genes horA and horC were originally found in L. brevis, but later they were also found in L. lindneri, L. paracollinoides and in the best known P. damnosus(Suzuki et al., 2006).

Curiously, HorA shows a resemblance of 54% to OmrA, a membrane transporter of Oenococcus oeni, related to the tolerance of this bacterium from wine to ethanol and other stressors (Bourdineaud et al 2004) (See some more about O. oeni in my post on the bacteria of the vine and the wine). Therefore, it is probable that HorA also has functions of exclusion of other compounds aside from those of the hops. It has been seen that these horAand horC resistance genes and their flanking regions are well preserved and have sequences almost identical to the different species that have them. Therefore, it is very likely that some have been acquired from others by means of horizontal gene transfer, by plasmids or transposons, as is usual in many other bacteria (Suzuki 2011).

As we see in Figure 9, protons are pumped out by an ATPase, and the consumption of ATPs is compensated by forming it thanks to the consumption of substrates such as citrate, malate, pyruvate or arginine. Another mechanism of resistance, passive in this case, is the modification of the composition of membrane fatty acids, with the addition of more saturated ones, such as C16:0, which reduces the membrane fluidity and makes it difficult the entrance of the hop compounds. This also reminds us of the changes in membrane of O. oeni related to the resistance to ethanol (Margalef-Català et al 2016). The cell wall also changes its composition in the presence of the hop alpha-iso-acids, increasing the amount of high molecular weight lipoteichoic acid, which would also be a barrier. We also see (Figure 9) how hop compounds can lower the intracellular levels of Mn2+, and then a greater synthesis of Mn-dependent proteins is observed, and a greater capture of Mn2+ from outside. Finally, cells of L. brevis reduce their size when they are in beer (Figure 10), probably in order to decrease the extracellular surface, thus minimizing the effect of external toxic compounds (Suzuki 2011).

All these mechanisms have been studied in L. brevis strains harmful to beer, but it is assumed that the resistance of beneficial bacteria from Lambic and others would be due to the same mechanisms, since they are of the same bacterial species.

As a conclusion to all said, we see that LAB have outstanding roles as beneficial in various aspects of brewery and malting, despite their most known role of harmful in the processing of the most common beers.

Lowe DP & Arendt EK (2004) The use and effects of lactic acid bacteria in malting and brewing with their relationships to antifungal activity, mycotoxins and gushing: a review. J Inst Brew 110, 163-180

At the same time it has been seen that probiotics can be a good solution for many diseases with affected gut microbiota. Indeed, the beneficial role of probiotics to reduce gastrointestinal inflammation and prevent colorectal cancer has been proven.

However, recently it has been found that probiotics may have beneficial effects in other parts of the body beyond the gastrointestinal tract, particularly with immunomodulatory effects on an hepatocellular carcinoma (HCC). In this way, researchers at the University of Hong Kong, along with other from University of Eastern Finland, have published a study (Li et al, PNAS, 2016), where they have seen reductions of 40% in weight and size of HCC liver tumours in mice which were administered with a new mixture of probiotics, “Prohep.”

Hepatocellular carcinoma (HCC) is the most common type of liver cancer is the 2nd most deadly cancers, and it is quite abundant in areas with high rates of hepatitis. In addition, sorafenib, the drug most widely used to reduce the proliferation of tumour, is very expensive. The cost of this multikinase inhibitor is €3400 for 112 tablets of 200 mg, the recommended treatment of four pills a day for a month. Instead, any treatment with probiotics that would proved to be effective and could replace this drug would be much cheaper.

In the mentioned work, Li et al. (2016) fed mice with Prohep for a week before inoculating them with a liver tumour, and observed a 40% reduction in tumour weight and size in comparison to control animals. As shown in Figure 1, the effect was significant at 35 days, and also for those who were given the Prohep the same day of tumour inoculation. Obviously, the effect of tumour reduction was much more evident when the antitumour compound Cisplatin was administered.

These researchers saw that tumour reduction was due to the inhibition of angiogenesis. This is the process that generates new blood vessels from existing ones, something essential for tumour growth. In relation to the tumour reduction, high levels of GLUT-1 + hypoxic were found. That meant that there was hypoxia caused by the lower blood flow to the tumour, since this was 54% lower in comparison to controls.

Figure 1. Change in tumour size. ProPre: administration of Prohep one week before tumour inoculation; ProTreat: administration of Prohep the same day of tumour inoculation; Cisplatin: administration of this antitumoral. (Fig 1B from Li et al, 2016).

These authors also determined that there was a smaller amount of pro-inflammatory angiogenic factor IL-17 and of Th17 cells of the immune system, cells also associated with cancer. The lower inflammation and angiogenesis could limit the tumour growth.

Moreover, these researchers established that the beneficial effects of probiotics administration were associated with the abundance of beneficial bacteria in the mice gut microbiota, analysed by metagenomics. So, probiotics modulate microbiota, favouring some gut bacteria, which produce anti-inflammatory metabolites such as cytokine IL-10 and which suppress the Th17 cell differentiation.

Some of the bacteria identified by metagenomics in the microbiota of mice that were administered with Prohep were Prevotella and Oscillibacter. The first is a bacteroidal, gram-negative bacterium, which is abundant in the microbiota of rural African child with diets rich in carbohydrates. Oscillibacter is a gram-positive clostridial, known in humans as a producer of the neurotransmitter GABA. Both are an example of the importance of some clostridial and bacteroidals in the gut microbiota. In fact, they are majority there, and although they are not used as probiotics, are found increasingly more positive functions, such as avoiding allergies (see “The good clostridia avoid us from allergies“).

It is known that these bacteria produce anti-inflammatory metabolites and therefore they would be the main involved in regulating the activity of immune cells that cause tumour growth. The observed reduction of tumour in these experiments with mice would be the result of combined effect of these administered probiotic bacteria together with the microbiota itself favoured by them. We see a potential outline of these actions in Figure 3.

Figure 3. Simplified diagram of the possible mechanisms of gut bacteria influencing on the polarization of Th17 cells in the lamina propria of the intestinal mucosa. The microbiota bacteria activate dendritic cells, which secrete cytokines (IL-22, IL-23, IL-27). The bacteria can promote Th17 immunity inducing IL-23, which can be involved by means of TLR ligands signal or extracellular ATP or serum amyloid A (SAA). Meanwhile, some probiotic strains could inhibit the development of Th17 by means of the production of IL-12 and IL-27, in addition to promoting the growth and colonization of the bacteria that induce Th17 (Sung et al 2012, Fig. 2).

Although we know that the cancer progression is a very complex process and that in the tumour microenvironments there is an infiltration of many different types of immune system cells, such as T cells, neutrophils, killer cells, macrophages etc, the Th17 helper cell subpopulation appears to be prevailing in the tumour progression, and therefore these effects of probiotics and microbiota open good prospects.

It is still early to say whether these findings will contribute to the treatment of human liver cancer, and therefore research in humans is needed, in order to see if these probiotics could be used as such or in tandem with some drug, depending on the tumour stage and size. In any case, all this opens a new range of possibilities for research of the molecular mechanisms of the beneficial effects of probiotics beyond the intestinal tract.

The vine growers believe that the land on which they grow vines gives the wines a unique quality, and that is called terroir. We can consider that the physiological response of the vines to the type of soil and climatic conditions, together with the characteristics of the variety and form of cultivation, result in a wine organoleptic properties that define their terroir (Zarraonaindia et al 2015 ). However, it is not known if there could be a very specific microbiota of each terroir, as this subject has been barely studied.

Wine microorganisms in the grapes? Saccharomyces is not there or it has not been found there

The main protagonists of wine fermentations, alcoholic one (yeast Saccharomyces cerevisiae) and malolactic one (lactic acid bacteria Oenococcus oeni) usually do not appear until the must grape is fermenting to wine, in the cellar. In normal healthy grapes, S. cerevisiae is hardly found.

Oenococcus oeni in the grapes ? We have found it !

Regarding O. oeni, so far very little has been published about its presence and isolation from the grapes. In some works, as Sieiro et al (1990), or more recently Bae et al (2006), some lactic acid bacteria (LAB) have been isolated from the surface of grapes, but not O. oeni. Only Garijo et al (2011) were able to isolate a colony (only one) of O. oeni from Rioja grapes. Moreover, DNA of O. oeni has been detected in a sample of grapes from Bordeaux (Renouf et al 2005, Renouf et al 2007) by PCR-DGGE of rpoB gene, although in these works no Oenococcus has been isolated.

I am pleased to mention that recently our team have managed to isolate O. oeni from grapes, and typify them, and we are now working on a publication about it (Franquès et al 2015). Indeed, our research team of lactic acid bacteria (BL-URV), together with colleagues working on yeasts from the same group “Oenological Biotechnology” (Faculty of Oenology at the Universitat Rovira i Virgili in Tarragona, Catalonia, Spain) is working on a European project, called “Wildwine “(FP7-SME-2012 -315065), which aims to analyse the autochthonous microorganisms of Priorat area (South Catalonia), and select strains with oenological potential. This project also involves the Priorat Appellation Council and the cellar Ferrer-Bobet, as well as research groups and associations wineries from Bordeaux, Piedmont and Greece. In the framework of this project we took samples of grapes (Grenache and Carignan) from several vineyards of Priorat (Figure 1), as well as samples of wines doing malolactic fermentation. From all them we got 1900 isolates of LAB. We optimized isolation from grapes from the pulp and juice with various methods of enrichment, and so we got 110 isolated bacteria from grapes, identified as O. oeni by specific molecular techniques. Once typified, we have found that the molecular profiles of these strains do not coincide with commercial strains and so they are autochthonous. In addition, some of these strains from grapes were also found in the corresponding wine cellars.

The grapes have a complex microbial ecology, including yeasts, mycelial fungi and bacteria. Some are found only in grapes, such as parasitic fungi and environmental bacteria, and others have the ability to survive and grow in wines: especially yeasts, lactic acid bacteria (LAB) and acetic acid bacteria. The proportion of all them depends on the maturation of the grapes and the availability of nutrients.

When the fruits are intact, the predominant microbiota are basidiomycetous yeasts as Cryptococcus and Rhodotorula, but when they are more mature, they begin to have micro fissures that facilitate the availability of nutrients and explain the predominance just before the harvest of slightly fermentative ascomycetes as Candida, Hanseniaspora, Metschnikowia and Pichia. When the skin is already damaged more damaging yeasts may appear, as Zygosaccharomyces and Torulaspora, and acetic acid bacteria. Among the filamentous fungi occasionally there may have some very harmful as Botrytis (bunch rot) or Aspergillus producing ochratoxin. Although they are active only in the vineyard, their products can affect wine quality.

On the other hand, environmentally ubiquitous bacteria have been isolated from the grapes skin, as various Enterobacteriaceae, Bacillus and Staphylococcus, but none of them can grow in wine (Barata et al 2012).

Coming back to the possible specific microbiota of terroir, it has been found that some volatile compounds contributing to the aroma of the wine, such as 2-methyl butanoic acid and 3-methyl butanol, are produced by microorganisms isolated in the vineyards, as Gram-positive bacterium Paenibacillus, or the basidiomycetous fungus Sporobolomyces or the ascomycetous Aureobasidium. Therefore, there could be a relationship between some of the microbial species found in grapes and some detected aromas in wine, coming from the must of course (Verginer et al 2010).

Metagenomics as analytical tool of microbiota from grapes

Since conventional methods of isolation and cultivation of microorganisms are slow, laborious and some microbes cannot be grown up in the usual isolation media, massive sequencing methods or metagenomics are currently used. These consist of analysing all the DNA of a sample, and deducing which are the present microorganisms by comparing the sequences found with those of the databases. For bacteria the amplified DNA of V4 fragment from 16S RNA gene is used (Caporaso et al 2012).

This technique has been used with samples of botrytized wines (Bokulich et al 2012) and various LAB have been found (but not Oenococcus), including some not normally associated with wine. It has also been used to see the resident microbiota in wineries and how it changes with the seasons, resulting that in the surfaces of tanks and machinery of the cellar there is a majority of microorganisms neither related with wine nor harmful (Bokulich et al 2013).

With this technique Bokulich et al (2014) have also analysed the grapes and they have seen clear differences between the proportions of bacterial groups (and fungi) from different places, different varieties, as well as environmental or bio geographical conditions. For example, when analysing 273 samples of grape musts from California, the 3 varieties (Cabernet, Chardonnay and Zinfandel) are quite discriminated in a principal components analysis with respect to the bacterial communities found in each sample (Figure 2).

Thus, the dominant bacterial taxa or groups in a variety or given environment could provide some specifics traits on those wines, and this could explain some regional or terroir patterns in the organoleptic properties of these wines (Bokulich et al 2014).

We have also carried out a massive sequencing study with the same grape samples from which we have obtained isolates of O. oeni, as said before (Franquès et al 2015), and in more than 600,000 analysed sequences of 16S rRNA, we have found mainly Proteobacteria and Firmicutes. Among these gram-positive, we have found sequences of lactic acid bacteria (15%) and from these we have successfully confirmed the presence of O. oeni in 5% of the sequences. Therefore, we have isolated O. oeni from grapes and we have detected their DNA in the samples.

The bacterial microbiota of the vineyards and soil

As we see, microbiota of grapes and wine has been studied a little, but the soil microbiota has not been characterized. This one can define more clearly the terroir, which is influenced by the local climate and characteristics of the vineyard.

In Figure 3 the main genera found in different parts of the vine and soil are summarized (Gilbert et al 2014).

Recently an interesting scientific work (Zarraonaindia et al 2015) has been published on this subject, with the aim to see if the soil could be the main original source of bacteria that colonize the grapes. These authors took samples of soil, roots, leaves, flowers and grapes from Merlot vines, from different areas and years, of Suffolk, New York, and they analysed the bacterial DNA by 16S rRNA sequencing. They found that 40% of the species found were present in all samples of soil and roots, while there was more variability in leaves and fruits, and moreover, 40% of those found in leaves and fruits were also found in soils. All this suggests that many bacteria originate in the soil.

Regarding the type of bacteria, they found that Proteobacteria (especially Pseudomonas and Methylobacterium) predominated (Figure 4), mainly in the aerial parts of the plant. There were also Firmicutes as expected, and Acidobacteria and Bacteroides.

Figure 4. Composition of the bacterial community, at Phylum level, in samples from different organs of the vine and its soil (Zarraonaindia et al 2015).

Although variations were observed in all samples depending on the year (there may be different climatic conditions) and according to different edaphic factors (pH, C: N, humidity), the principal-components analysis (Figure 5) showed that the main types of samples (soil, roots, leaves, grapes) differ quite well, and bacterial taxon composition in samples of grape juice before fermentation is similar to that of grapes.

This suggests that the bacterial community found in grapes remains relatively stable until the processing to musts, and that it is more stable than the differences between organs. At the same time, a large number of representatives of bacterial phyla of the grapes come from the soil. This can be explained because when grapes are harvested by hand, they are often placed in boxes that are left on the ground, or for mechanical harvest, the machinery used removes the soil and generates dust, which can colonize the grapes.

Therefore, the soil microbiota is a source of bacteria associated with vines and may play a role in the must and therefore in the wine, and potentially in the formation of the terroir characteristics. Some of these bacteria may have some roles not yet known in productivity or disease resistance of the plant, or contribute to the organoleptic characteristics of wine (Zarraonaindia et al 2015).

In addition, and thinking in wine microorganisms responsible for fermentations, as said, in our laboratory we have confirmed that there are some O. oeni strains in grapes and we have confirmed this by detecting their DNA in the same grapes.

It seems to be so: the microbes in our gastrointestinal tract (GIT) influence our choice of food. No wonder: microbes, primarily bacteria, are present in significant amounts in GIT, more than 10 bacterial cells for each of our cells, a total of 1014 (The human body has about 1013 cells). This amounts to about 1-1.5 kg. And these bacteria have lived with us always, since all mammals have them. So, they have evolved with our ancestors and therefore they are well suited to our internal environment. Being our bodies their habitat, much the better if they can control what reaches the intestine. And how can they do? Then giving orders to the brain to eat such a thing or that other, appropriate for them, the microbes.

Well, gone seriously, there is some previous work in this direction. It seems there is a relationship between preferences for a particular diet and microbial composition of GIT (Norris et al 2013). In fact, it is a two-way interaction, one of the many aspects of symbiotic mutualism between us and our microbiota (Dethlefsen et al 2007).

There is much evidence that diet influences the microbiota. One of the most striking examples is that African children fed almost exclusively in sorghum have more cellulolytic microbes than other children (De Filippo et al 2010).

The brain can also indirectly influence the gut microbiota by changes in intestinal motility, secretion and permeability, or directly releasing specific molecules to the gut digestive lumen from the sub epithelial cells (neurons or from the immune system) (Rhee et al 2009).

The GIT is a complex ecosystem where different species of bacteria and other microorganisms must compete and cooperate among themselves and with the host cells. The food ingested by the host (human or other mammal) is an important factor in the continuous selection of these microbes and the nature of food is often determined by the preferences of the host. Those bacteria that are able to manipulate these preferences will have advantages over those that are not (Norris et al 2013).

Recently Alcock et al (2014) have reviewed the evidences of all this. Microbes can manipulate the feeding behaviour of the host in their own benefit through various possible strategies. We’ll see some examples in relation to the scheme of Figure 2.

Figure 2. As if microbes were puppeteers and we humans were the puppets, microbes can control what we eat by a number of marked mechanisms. Adapted from Alcock et al 2014.

People who have “desires” of chocolate have different microbial metabolites in urine from people indifferent to chocolate, despite having the same diet.

Dysphoria, id est, human discomfort until we eat food which improve microbial “welfare”, may be due to the expression of bacterial virulence genes and perception of pain by the host. This is because the production of toxins is often triggered by a low concentration of nutrients limiting growth. The detection of sugars and other nutrients regulates virulence and growth of various microbes. These directly injure the intestinal epithelium when nutrients are absent. According to this hypothesis, it has been shown that bacterial virulence proteins activate pain receptors. It has been shown that fasting in mice increases the perception of pain by a mechanism of vagal nerve.

Microbes can also alter food preferences of guests changing the expression of taste receptors on the host. In this sense, for instance germ-free mice prefer more sweet food and have a greater number of sweet receptors on the tongue and intestine that mice with a normal microbiota.

The feeding behaviour of the host can also be manipulated by microbes through the nervous system, through the vagus nerve, which connects the 100 million neurons of the enteric nervous system from the gut to the brain via the medulla. Enteric nerves have receptors that react to the presence of certain bacteria and bacterial metabolites such as short chain fatty acids. The vagus nerve regulates eating behaviour and body weight. It has been seen that the activity of the vagus nerve of rats stimulated with norepinephrine causes that they keep eating despite being satiated. This suggests that GIT microbes produce neurotransmitters that can contribute to overeating.

Neurotransmitters produced by microbes are analogue compounds to mammalian hormones related to mood and behaviour. More than 50% of dopamine and most of serotonin in the body have an intestinal origin. Many persistent and transient inhabitants of the gut, including E. coli, several Bacillus, Staphylococcus and Proteus secrete dopamine. In Table 1 we can see the various neurotransmitters produced by GIT microbes. At the same time, it is known that host enzymes such as amine oxidase can degrade neurotransmitters produced by microorganisms, which demonstrates the evolutionary interactions between microbes and hosts.

Table 1. Diversity of neurotransmitters isolated from several microbial species (Roschchina 2010)

Neurotransmitter

Genera

GABA (gamma-amino-butyric acid)

Lactobacillus, Bifidobacterium

Norepinephrine

Escherichia, Bacillus, Saccharomyces

Serotonin

Candida, Streptococcus, Escherichia, Enterococcus

Dopamine

Bacillus, Serratia

Acetylcholine

Lactobacillus

Some bacteria induce hosts to provide their favourite nutrients. For example, Bacteroides thetaiotaomicron inhabits the intestinal mucus, where it feeds on oligosaccharides secreted by goblet cells of the intestine, and this bacterium induces its host mammal to increase the secretion of these oligosaccharides. Instead, Faecalibacterium prausnitzii, a not degrading mucus, which is associated with B. thetaiotaomicron, inhibits the mucus production. Therefore, this is an ecosystem with multiple agents that interact with each other and with the host.

As microbiota is easily manipulated by prebiotics, probiotics, antibiotics, faecal transplants, and changes in diet, controlling and altering our microbiota provides a viable method to the otherwise insoluble problems of obesity and poor diet.

Breast milk, besides being very nutritious, provides bioactive constituents that favor the development of the infant immune system and prevent diseases. From this point of view, the best known compounds are maternal immunoglobulins, immunocompetent cells and various antimicrobials. It also contains prebiotic substances, ie, several molecules such as oligosaccharides, which stimulate the growth of specific bacteria in the gut of the child.

However, other important constituents of breast milk, unsuspected until few years ago, are the bacteria. In fact, milk is not sterile, it contains microorganisms, primarily beneficial bacteria that help to establish the intestinal microbiota of the newborn, and which are the first to settle there. Although artificial milk are made to resemble the breast milk, they remain distinct and do not contain bacteria. And for this reason, the intestinal microbiota of breast-fed infants is different than those fed with artificial breast milk.

Just a few weeks ago was published a work ( Cabrera-Rubio et al., 2012 ) in the American Journal of Clinical Nutrition that had a good coverage in media, blogs and networks ( click here for an example), because it shows the great diversity of bacteria present in the breast milk.

Although this work done by Valencian researchers (Cavanilles Institute, University of Valencia and CSIC-IATA) with Finnish researchers is not the first study that examines this issue, this study shows that bacteria are from very diverse species.

One of the novelties of this paper is the method used, taking advantage of the latest molecular biology: they studied the microbiome in breast milk, that is, the analysis of all possible bacteria present in the samples, by DNA sequencing, without the traditional isolation of living bacteria in plates. To do so, from the aseptically collected milk, DNA is extracted and the gene fragments of bacterial 16S rRNA are amplified by PCR. These amplified genes are sequenced by pyrosequencing (454 Roche GS-FLX), the most innovative and rapid sequencing technology: a machine of this allows about 400 million base pairs (bp) of DNA in 10 hours. From the rRNA gene of each possible bacteria some 500 bp are sequenced. Thus, in this study about 120,000 sequences have been analyzed, corresponding to 2600 sequences per milk sample.

By comparing these sequences with the databases and applying statistical methods conclusions can be drawn on what taxonomic groups (genera and species) bacteria are present and in what proportion.

As shown in the figure above, Cabrera et al. found in the milk of healthy mothers that the predominant genera are Leuconostoc, Weissella, Lactococcus and Staphylococcus, of which the first three are lactic acid bacteria. Although these are predominant in colostrum and milk during the first months, then other bacteria are increasing their numbers, such as Veillonella Leptotrichia (anaerobic gram-negative bacteria), which are typical commensal of the oral cavity. In total, about 1000 species have been found, that vary depending on the mother. Curiously, there are significant variations on whether delivery had been vaginal or cesarean, and on the obesity of the mother. The reasons for this are not yet clear.

And where the bacteria in breast milk come from ?

Besides the identifications made in this study of Cabrera et al. (2012) on the basis of DNA present, it has been observed by making viable counts that the total number of bacteria in breast milk is between 2·104 and 3·105 per ml (Juan Miguel Rodríguez), that is, a quantity not negligible . What is its origin?

The study of the microbiome of Cabrera et al. also concluded that the composition of different bacteria is somewhat different from that of other bacterial communities in the human body (the human bacterial niches: skin, mouth, digestive system, vagina, etc), and therefore the milk microbiome is not a particular subset of one of these niches.

As discussed in a recent review published by this group (Fernández et al 2012), the bacteria present in the breast milk would come from three possible sources (figure below): skin bacteria from the same breast, the oral cavity of the infant, and the most surprising, commensal bacteria of the maternal gut that pass to milk by the entero-mammary pathway.

Potential sources of bacteria present in human colostrum and milk, including the transit of intestinal commensal bacteria to the milk by the entero-mammary pathway (Fernández et al., 2012).DC: dendritic cells.

Indeed, several studies had shown that dendritic cells cross the intestinal epithelium (between enterocytes) and may take commensal bacteria of the gut lumen, incorporating them by endocytosis, but keeping them alive. See details in the following diagram.

These dendritic cells travel through the circulatory system, reaching the mammary glands, where it seems that include bacteria to milk. This is the the entero-mammary pathway.

In this breast microbiota, bacteria from breast skin and from oral cavity of the child also would be incorporated. Some of these bacteria the child’s oral cavity are actually related to those of its gastrointestinal tract. As the first bacteria inhabiting this tract are those of the vaginal microbiota during birth (and intestinal if delivery is cesarean), this would explain the phylogeny of certain bacteria in the milk of these microbiota.

In summary, we see as the “good” bacteria (lactic acid bacteria, but also bifidobacteria and other) from maternal gut, by different ways, arrive to breast milk, and the reach the child’s gut, developing there the child’s microbiota, and helping to complete the neonatal immune system.